Podcasts about lptcs

In arthropods like Drosophila, Down syndrome cell adhesion molecules (Dscam1) exhibit enormous molecular diversity. A single Dscam1 gene encodes a large superfamily of neuronal cell recognition proteins that control neuronal outgrowth and anatomy. A comparable function is exhibited by the vertebrates DSCAMs of which only few isoforms exist. However, it is largely unknown, if and how this function of Dscams affects neuronal function and the control of behavior by the nervous system. In this thesis, I employed an arsenal of genetic techniques to perturb the expression level of Dscam1 isoforms in directionally selective Lobula Plate Tangential Cells (LPTCs). LPTCs of the Vertical (VS) and the Horizontal System (HS) were chosen as a model system because of their well-documented anatomy, role in information processing and behavior. Though, only little is known about the developmental mechanisms and molecular factors controlling the morphogenesis and wiring of these cells. The central aim of my study thus is to reveal a possible role of Dscam1 in the growth and development of the complex dendrites of in particular HS cells. Furthermore, my work aims at establishing a novel model system for integrated studies on the development and function of LPTCs by genetic manipulations of Dscam1 expression. My results demonstrate that Dscam1 is expressed broadly in the fly visual system including HS-cells (immunolabeling of the conserved intracellular domain). Loss of Dscam1 function and reduced isoform diversity consistently elicited misrouting and self-crossings of neurites in LPTC dendrites. In contrast, misexpression of selected single Dscam1 isoforms caused a severe reduction in the size and branching complexity of LPTC dendrites. The dendritic gain-of-function phenotype (ectopic expression of the Dscam1 isoform 11.31.25.1) was strongly dependent on the time of onset of misexpression during development. These results demonstrate that Dscam1 contributes to the development of LPTC dendrites. This system can now be used to (A) address a possible role of Dscam1 in the function of neurons and circuitries and (B) to address the interplay of anatomy and function of LPTC dendrites. In further side projects I aimed at the development of additional genetic tools for the investigation of the role of LPTCs in behavior and for studies on the wiring of LPTCs to the presynaptic circuitry. I established a heat-shock protocol for the ablation of specified LPTCs by RicinA expression and I generated a fly line for the expression of TN-XXL (a genetically encoded calcium biosensor) in small cell clusters or individual cells. Finally, I participated in efforts to establish a virus based retrograde labeling method in Drosophila.

Dendrite morphology is the most prominent feature of nerve cells, investigated since the origins of modern neuroscience. The last century of neuroanatomical research has revealed an overwhelming diversity of different dendritic shapes and complexities. Its great variability, however, largely interferes with understanding the underlying principles of neuronal wiring and its functional implications. This work addresses this issue by studying a morphological and functional exception- ally conserved network of neurons located in the visual system of flies. Lobula Plate Tangential Cells (LPTCs) have been shown to compute motion vision and contribute to the impressive flight capabilities of flies. Cells of this system exhibit a high degree of constancy in topographic location, morphology and function over all individuals of one species. This constancy allows investigation of functionally identical cells over a large population of flies, and therefore potentially to truly understand the underlying principles of their morphologies. Supported by a large database of in vivo cell reconstructions and a computational quantification framework, it was possible to uncover some of those principles of LPTC anatomy. We show that the key to the cells’ morphological identity lies in the size and shape of the area they span into. Their detailed branching structure and topology is then merely a result of a common growth program shared by all analyzed cells. Application of a previously published branching theory confirmed this finding. When grown into the spanning fields obtained from the in vivo cell reconstruction, artificial cells could be synthesized that resembled all anatomical properties that characterize their natural counterparts. Furthermore, the morphological comparison of the same identified cells in Calliphora and Drosophila allowed to study a functionally conserved system under the influence of extensive down-scaling. The huge size reduction did not affect the underlying branching principles: Drosophila LPTCs followed the very same rules as their Calliphora coun- terparts. On the other hand, we observed significant differences in complexity and relative diameter scaling. An electrotonic analysis revealed that these differences can be explained by a common functional architecture implemented in the LPTCs of both species. Finally, we could modify the LPTC neuronal interaction behavior thanks to the genetical accessibility of Drosophila’s wiring program. The transmembrane protein family Dscam has been shown to mediate the process of adhesion and repulsion of neurites. By manipulating the molecular Dscam profile in Drosophila LPTCs it was possible to change their morphological expansion. The low variability of the LPTCs spanning field in wild type flies and their two-dimensional extension allowed to thoroughly map these morphological alterations in flies with Dscam modifications. In line with the LPTCs retinotopic input arrangement, electrophysiological experiments yielded an inherent linear relationship of their locally reduced dendritic coverage and their locally reduced stimulus sensitivity. With this work I hope to contribute to the general understanding of neuronal morphology of LPTCs and to present a valuable workflow for the analysis of neuronal structure.

Visual motion detection is of major importance for flies as they use the optic flow generated by their self-motion to control their course during flight. This so called optomotor behavior is thought to be controlled by a set of large-field motion-sensitive cells in the optic lobes called lobula plate tangential cells (LPTCs). LPTCs come in different variants and are tuned to different preferred directions. Their responses can be explained by assuming input from an array of local motion detectors of the correlation-type. In addition, they receive input from other LPTCs from both the ipsi- and the contralateral hemisphere. Response properties of LPTCs have been extensively described in large fly species. However, information about the presynaptic circuits that constitute the local motion detectors is still largely missing. Research on the fruit fly Drosophila promises to close this gap as it allows for combining physiological recordings from motion-sensitive cells with a genetic manipulation of the system. In that way the function of neurons too small for electrophysiological recordings can also be analyzed. Here, I provide important steps towards elucidating the cellular implementation of the correlation-type motion detector in the fly brain. First, I tested different genetically encoded Calcium indicators (GECIs) expressed in LPTCs by stimulating the neurons with potassium chloride. These experiments revealed that GECIs are functional in LPTCs and might thus be useful for monitoring neuronal activity in the visual system. Second, I described the response properties of HS (horizontal system) cells, a prominent subgroup of LPTCs in Drosophila. There are three HS cells per hemisphere, HSN, HSE and HSS. All of them are tuned to horizontal motion in a directionally selective way. I could show that their responses are indicative of correlation-type motion detectors providing input to them. In addition, they receive information from the contralateral side most likely via other LPTCs. HS cells not only have strongly overlapping dendritic trees in the lobula plate accounting for their large and overlapping receptive fields, but are also coupled electrically with each other. Extensive electrical connections can also be found to descending neurons in their output region in the central brain. This characterization of HS cells is important for two reasons: i) Their responses can be used as a read-out for the effects of manipulating the presynaptic motion detection circuitry in the fly by genetic techniques; ii) they can be correlated with behavioral reactions induced by horizontal motion to study how optomotor responses are controlled in the fly. Third, I studied the input pathways to the LPTCs in the lamina, the first optic neuropile after the compound eye. From all lamina cells, L1 and L2 are the most prominent neurons and were previously shown to provide the major input to the motion detection circuits. By genetically restoring synaptic input to either one of the two pathways I revealed that these two types of cells indeed provide the major input to LPTCs. However, their functional specialization for light increments and light decrements, disclosed by blocking their synaptic output, could not be revealed in these experiments. As L1 and L2 turned out to be electrically coupled with each other restoring the input to only one cell type also restores the input to the other one. Finally, I analyzed response properties of HS cells whose dendritic structure has been altered by overexpression of Dscam (Down syndrome cell adhesion molecule) during development. Dscam is a protein that comes in a large number of different isoforms and is thought to play a major role in self-recognition and thus proper dendritic and axonal branching. HS cells that misexpress a single isoform develop smaller and less overlapping dendritic trees in the lobula plate. These anatomical defects are accompanied by smaller receptive fields but otherwise normal motion responses. All these experiments show that the combination of physiological and genetic tools is a promising approach for dissecting neural circuits and gaining new insights into information processing in the brain. Continuation of this approach will hopefully bridge the gap between neurons of the lamina and the lobula plate by revealing the local motion detectors in the intermediate neuropile, the medulla.

In the past few decades, the lobula plate of the fly has emerged as one of the leading models for the neural processing of optic flow stimuli that give rise to visual orientation behaviors (for recent reviews see Borst and Haag, 2002; Egelhaaf et al., 2002; Egelhaaf et al., 2002; Borst and Haag, 2007). The relative simplicity and accessibility of this neural system allows researchers to characterize the neural mechanisms that are thought to link the visual stimuli and the resulting behavioral responses. In the lobula plate, a set of 60 motion sensitive lobula plate tangential cells (LPTCs) integrate visual motion information from an array of local motion detectors, which form a retinotopic map of the fly’s visual space in the lobula plate. The selective pooling of local, direction selective inputs, together with a network of unilateral and bilateral interactions between LPTCs, shape and tune the response properties of LPTCs to behaviorally relevant optic flow stimuli. Over the years, lobula plate researchers assembled a formidable array of measurement and perturbation techniques that are usually available only in in-vitro systems. Additionally, the lobula plate and its presynaptic circuitry have been the subject of extensive and detailed modeling which allows a deeper synthetic understanding of the empirical results, as well as a more efficient and detailed way to generate hypotheses. In this work I used a selection of these tools to explore the role of intracellular processing of visual motion information in lobula plate neurons and the significance of spatial segregation and aggregation of these cells’ inputs in the context of their sensory function. Previous work on a network of ten LPTCs of the vertical system (VS cells) resulted in a prediction that due to lateral, gap-junction coupling of neighboring VS cells in their axon-terminals, the receptive fields of these cells should be broader in the axonal region than in the dendritic regions. I tested and confirmed this prediction using in-vivo calcium imaging and intracellular recordings. Using single-electrode voltage clamp I was able to perturb the flow of information in these cells and isolate the source of input responsible for this broadening, confirming that the coupling indeed takes place in the axon terminal. The separation of feed-forward, synaptic input in the dendrites from lateral, gap-junction coupling in the axon-terminals allowed me to experimentally ask what is the function of the receptive field broadening. Relying on model predictions, I showed that this broadening results in a more stable and smooth representation of optic flow in the output region of the cells than in their input region, when the fly is presented with naturalistic, patchy and non-uniform stimuli. I then showed, using a simplified compartmental model that the separation of axonal gap-junctions from the dendritic synaptic input makes the gap-junction coupling more effective, and is thus necessary to ensure the functionality of the lateral interactions.

The development of dendrites leads to the establishment of cell-type specific morphology of dendritic trees that eventually determines the way in which synaptic information is processed within the nervous system. The aim of this study was to investigate dendritogenesis of Drosophila motion-sensitive Lobula Plate Tangential Cells (LPTCs) and to understand the role of cytoskeletal molecules in these developmental processes. I employed genetic techniques to obtain fluorescent labeling exclusively in the neurons of interest. In order to visualize the LPTCs confocal imaging was applied. Time point analysis allowed me to follow and describe the phases of LPTC differentiation in the intact Drosophila brain starting from the third instar larva throughout the pupal stages until adulthood. I determined the time when the initial growth of LPTC dendrites starts and showed it to be directional from the beginning. Additionally, I demonstrated that the phase of extensive dendritic growth and branching precedes reorganization processes that lead to establishment of the final architecture of LPTC dendritic trees. In parallel, I attempted to analyze the contribution of actin and tubulin in the shaping of the neurons. In these experiments actin-GFP localized to dendritic termini whereas tubulin-GFP was mainly observed in the primary dendritic branches. These data showed clear similarities between the cytoskeletal organization of LPTCs dendrites and vertebrate neurons. The discovery of the actin enrichment in dendritic termini made me conduct a set of experiments to test if these protrusions are the counterparts of vertebrate spines. I performed a thorough quantitative analysis of spine- like protrusions present on LPTC dendrites. Morphological features like the density and shape of the LPTC spine- like protrusions appeared to be comparable to hippocampal spines. Using immunohistochemical methods I demonstrated that LPTC spine-like protrusions are sites of synaptic contacts. The ultrastructural analysis supported the immunohistochemical data and showed that synaptic transmission takes place at the LPTC spine-like protrusions. Next, I tried to genetically modify these structures by generating LPTC mutant for genes which have vertebrate homologues known to alter spine morphology. I showed that dRac1 can modulate significantly the LPTC spine-like structure density. Finally, I tried to check if Drosophila LPTC spine-like structures are motile. To conclude, I showed an initial description of LPTC dendritogenesis and the subcellular localization of actin and tubulin in these neurons. The actin enriched spine-like structures detected on the LPTC dendrites are sites of synaptic contacts, thus resemble vertebrate spines.

As a fly flies around in the world the visual scene moves constantly across its eyes. Depending on its path, this elicits a particular large-field motion pattern called ‘flow field’. Since the flow-fields are characteristic for particular flight trajectories they can be used to guide behavior, in particular to control the course of the fly. In the blowfly, these visual motion cues are mediated by a set of 60 motion-sensitive neurons called lobula plate tangential cells (LPTCs). The directionally selective response of the LPTCs has been ascribed to the integration of local motion information across their extensive dendritic trees. As the lobula plate is organized retinotopically the receptive fields of the tangential cells ought to be determined by their dendritic architecture. This appears not always to be the case. Recent experiments have revealed many lateral connections among tangential cells that appear to mediate their often complex receptive fields. Here single cells were ablated in order to determine which lateral connections are functionally important. I found that the ablation of a single cell, or class of cells revealed that the lateral connections among LPTCs can be the source of their local motion input, or augment the feedfoward input from local motion elements through either dendro-dendritic and axonal-axonal connections. Other connections between LPTCs were found to have no discernable functional significance and suggest that the lobula plate circuitry is yet to be fully revealed. The specific projects are outlined below. Input Circuitry to the HS- and CH-cells A single class of the lobula plate tangential cells the CH (centrifugal horizontal) neurons, play an important role in two pathways: figure-ground discrimination and flow-field selectivity. As was recently found, the dendrites of CH-cells are electrically coupled to the dendritic tree of another class of neurons sensitive to horizontal image motion, the horizontal system cells (HS). However, whether motion information arrives independently at both of these cells or is passed from one to the other is not known. Here I examine the ipsilateral input circuitry to HS and CH neurons by selective laser ablation of individual interneurons. I find that the response of CH neurons to motion presented in front of the ipsilateral eye is entirely abolished after the ablation of HS-cells. In contrast the motion response of HS-cells persists after the ablation of CH-cells. I conclude that HS-cells receive direct motion input from local motion elements, whereas CH-cells do not; their motion response is driven by HS-cells. This connection scheme is discussed with reference as to how the dendritic networks involved in figure-ground detection and flow-field selectivity might operate. Rotational Flow-Field Selectivity The group of neurons that processes horizontal motion forms a symmetric bilateral network that is able to combine information about motion presented in front of both eyes. Here I consider a group of 16 neurons whose connections have been explicitly identified. Each of these neurons has a large dendritic tree receiving information about ipsilateral local motion events that is spatially pooled to produce a directionally selective response. In addition, some of the lobula plate neurons are also sensitive to motion cues in front of the other eye. This information is carried by the spiking neurons H1, H2 and Hu that send their axons to the other side of the brain, where the H1- and H2-cells synapse onto 2 of the 3 HS-cells, and all three contralaterally projecting cell provide input to both CH-cells. The CH-cells are known to provide inhibitory input to the H1- and H2-cells. These network interactions appear to amplify the response to rotational stimuli and reduce the response to translation. I ablate either single HS-cells or both CH-cells in order to break the path whereby information about the opposite eye reaches the H1- and H2-cell. I did not find that these ablations affected the flow-field selectivity of either H1- and H2-cells. Network modeling showed that although the described circuitry does support rotational flow-field selectivity for the HS- and CH-cells, the model H2-cell does not show the expected flow-field selectivity. This suggests that the circuitry or cellular mechanisms underlying the response properties of the H2-cell are not completely understood. Basis of the Broad Receptive Field of VS-cells As the lobula plate is organized retinotopically the receptive fields of the tangential cells ought to be determined by their dendritic architecture. This appears not always to be the case. One compelling example is the exceptionally wide receptive fields of the vertical system (VS) tangential cells. Using dual intracellular recordings Haag and Borst (2004) found VS-cells to be mutually coupled in such a way that each VS-cell is connected exclusively to its immediate neighbours. This coupling may form the basis of the broad receptive fields of VS-cells. Here I tested this hypothesis directly by photo-ablating individual VS-cells. The receptive field width of VS-cells indeed narrowed after the ablation of single VS-cells, specifically depending on whether the receptive field of the ablated cell was more frontal or more posterior to the recorded cell. In particular, the responses changed as if the neuron lost access to visual information from the ablated neuron and those VS-cells more distal than it from the recorded neuron. These experiments provide compelling evidence that the lateral connections amongst VS-cells are a crucial component in the mechanism underlying their complex receptive fields, augmenting the direct columnar input to their dendrites. Vertical-Horizontal Interactions Two heterolaterally spiking cells, the H1- and H2-cells have been shown to be sensitive to vertical motion presented in the frontal portion of their receptive fields. Receptive field measurements performed here show that the H1-, VS1- and VS2-cells all respond to vertical downward motion across an almost completely overlapping portion of the frontal visual field. Using dual intracellular recordings Haag and Borst (2003) demonstrated that the VS1-cell but not the VS2-cell supplies input to both these cells. Through current injections into different compartments of the VS1-and VS2-cells I have provided physiological evidence that the output of VS1-cell near its dendritic arbors is the likely site of its input to the H1-cell. This coupling may form the basis of the vertical sensitivity of the H1- and H2-cell. I tested this hypothesis directly by recording the sensitivity of the H1-cell to horizontal and vertical motion in the frontal visual field both before and after the ablation of single VS1-cells. After the ablation of the VS1-cell the response of the H1-cell to vertical motion disappeared but its response to horizontal motion remained robust. These experiments demonstrate that the VS1-cell provides the input to the H1-cell that makes it sensitive to vertical motion in the frontal visual field likely through connections in their dendritic trees.